Long-term, non-invasive FTIR detection of low-dose ionizing radiation exposure.

Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.

Chromohalobacter salixigens Uronate Dehydrogenase: Directed Evolution for Improved Thermal Stability and Mutant CsUDH-inc X-ray Crystal Structure.

Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolution using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family library contained in total 16 mutations, only some of which when examined individually appreciably increased thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isolation, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t 0.5 +18 °C, such that k cat was unaffected by incubation for 1 hr at ~70 °C. X-ray crystal structure of CsUDH-inc showed tight packing of the mutated residue side-chains, and comparison of rescaled B-values showed no obvious differences between wild type and mutant structures. Activity of CsUDH-inc was severely depressed on glucuronic and galacturonic acids. Combining select combinations of only three mutations resulted in good or comparable activity on these uronic acids, while maintaining some improved thermostability with ΔK t 0.5 ~+ 10 °C, indicating potential to further thermally optimize CsUDH for hyperthermophilic reaction environments.

Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

Vaccines train the immune system to recognize a specific virus or bacterium so that the body can be better prepared against these harmful agents. To do so, many vaccines contain viral molecules called glycoproteins, which are specific to each type of virus. Glycoproteins that sit at the surface of the virus can act as 'keys' that recognize and unlock the cells of certain organisms, leading to viral infection. To ensure a stronger immune response, glycoproteins in vaccines are often arranged on a protein scaffolding which can mimic the shape of the virus of interest and trigger a strong immune response. Many scaffoldings, however, are currently made from natural proteins which cannot always display viral glycoproteins. Here, Ueda, Antanasijevic et al. developed a method that allows for the design of artificial proteins which can serve as scaffolding for viral glycoproteins. This approach was tested using three viruses: influenza, HIV, and RSV ­ a virus responsible for bronchiolitis. The experiments showed that in each case, the relevant viral glycoproteins could attach themselves to the scaffolding. These structures could then assemble themselves into vaccine particles with predicted geometrical shapes, which mimicked the virus and maximized the response from the immune system. Designing artificial scaffolding for viral glycoproteins gives greater control over vaccine design, allowing scientists to manipulate the shape of vaccine particles and test the impact on the immune response. Ultimately, the approach developed by Ueda, Antanasijevic et al. could lead to vaccines that are more efficient and protective, including against viruses for which there is currently no suitable scaffolding.

Minimally disruptive optical control of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Evolutionarily Conserved Allosteric Communication in Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevant-and, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase 1B (PTP1B) with bioinformatic analyses of the PTP family to examine allosteric communication in this class of enzymes. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP1B possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from both kinetic studies and mutational analyses show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation.

Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.

Resonant Soft X-Ray Scattering Provides Protein Structure with Chemical Specificity.

We introduce resonant soft X-ray scattering (RSoXS) as an approach to study the structure of proteins and other biological molecules in solution. Scattering contrast calculations suggest that RSoXS has comparable or even higher sensitivity than hard X-ray scattering because of contrast generated at the absorption edges of constituent elements, such as carbon and oxygen. Here, we demonstrate that working near the carbon edge reveals the envelope function of bovine serum albumin, using scattering volumes of 10-5 µL that are multiple orders of magnitude lower than traditional scattering experiments. Furthermore, tuning the X-ray energy within the carbon absorption edge provides different signatures of the size and shape of the protein by revealing the density of different types of bonding motifs within the protein. The combination of chemical specificity, smaller sample size, and enhanced X-ray contrast will propel RSoXS as a complementary tool to existing techniques for the study of biomolecular structure.

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses.

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.

Reconstruction from limited single-particle diffraction data via simultaneous determination of state, orientation, intensity, and phase.

Free-electron lasers now have the ability to collect X-ray diffraction patterns from individual molecules; however, each sample is delivered at unknown orientation and may be in one of several conformational states, each with a different molecular structure. Hit rates are often low, typically around 0.1%, limiting the number of useful images that can be collected. Determining accurate structural information requires classifying and orienting each image, accurately assembling them into a 3D diffraction intensity function, and determining missing phase information. Additionally, single particles typically scatter very few photons, leading to high image noise levels. We develop a multitiered iterative phasing algorithm to reconstruct structural information from single-particle diffraction data by simultaneously determining the states, orientations, intensities, phases, and underlying structure in a single iterative procedure. We leverage real-space constraints on the structure to help guide optimization and reconstruct underlying structure from very few images with excellent global convergence properties. We show that this approach can determine structural resolution beyond what is suggested by standard Shannon sampling arguments for ideal images and is also robust to noise.